The broad, long-term objective of this project is to establish and characterize the mechanism of action of ethanol on the GABAA receptor. The work will progress in three principal directions. First, we will establish the nature of interactions between ethanol and neuroactive steroids on the alpha1beta2gamma2 GABAA receptor. In these experiments, we will determine the range of endogenous steroids that can interact with the receptor to produce ethanol-mediated potentiation, and carry put experiments to gain insight into the molecular and kinetic mechanisms of ethanol action by using neuroactive steroids of varying effects and site-directed mutagenesis of the GABAA receptor subunits. Second, we will examine the mechanism of direct potentiating effect of ethanol (i.e., in the absence of a cofactor) on GABA activated currents from alpha4beta2delta receptors. And third, we will establish the potentiating effect of ethanol on native receptors in rodent brain slices. The specific goals of the proposed research are: (1) To establish whether endogenous neuroactive steroids and ethanol positively interact on the recombinant alpha1beta2gamma2 GABAA receptor to potentiate the receptor function. (2) To study the molecular and kinetic mechanisms of alpha1beta2gamma2 GABAA receptor modulation by ethanol. (3) To determine the mechanism of direct action of ethanol on recombinant alpha4beta2delta GABAA receptors. (4) To study the effect of ethanol on whole-cell currents in brain slices. The work will be carried out on recombinant wild type and mutant alpha1beta2gamma2 and alpha4beta-delta subunit-containing) GABAA receptors transiently expressed in HEK 293 cells, and on thalamic slices expressing native GABAA receptors. Receptor activation and its modulation by steroids and ethanol will be examined from the recordings of single-channel and whole-cell currents. The proposed research has been designed to test the hypothesis that ethanol-linked inebriation is mediated by the activation of the GABAA receptor in the central nervous system, and that receptor interaction with endogenous neuroactive steroids is a critical component in such activation of the GABAA receptor system. The results from the experiments will be used to examine the mechanism and structural requirements in the ethanol-mediated modulation of the GABAA receptor function.